Approval of Photofrin for use in Photodynamic Therapy (PDT) of esophageal cancer provides impetus for development of optimal treatment regimens. Investigations from our laboratories have utilized numerous experimental models, including cultured cells, multicell spheroids, tumors in vivo and the in vivo-in vitro paradigm, to determine discrete sites of action of porphyrin-induced photosensitization. We determined that significant inhibition of inner mitochondrial enzymes led to marked reduction in ATP levels in cells and in tumors in situ, preceding tumor regression. By modeling oxygen diffusion and its photochemical consumption, irradiation schemes were devised that markedly improved tumor response. These models will be used to investigate, and then increase, the efficacy of delta- aminolevulinic acid (delta-ALA) induced photosensitization, a conceptually different approach to PDT. Delta-ALA induces the endogenous formation of the photosensitizer protoporphyrin IX (PPIX) via the heme biosynthetic pathway. We propose to determine localization of sensitization by measurement of site-specific enzyme response, alterations in components of the heme biosynthetic pathway and test for rate-limiting steps by using molecular biology techniques to transfect and overexpress selected enzymes. Multicell spheroids will provide a model to analyze spatial and temporal PPIX synthesis as it relates to metabolic status. Towards the goal of optimization of PDT, a stepped irradiation scheme, as well as irradiation of the tumor bed, will be applied to neoplasms in vivo. Mechanistic studies of vascular and metabolic responses to PDT in real time will be pursued in vivo using MR imaging and spectroscopy. Response of primary mammary tumors to PDT will determine whether lesions in situ differ in sensitivity from transplanted tumors. Results from these experiments will provide insight into discrete actions of delta-ALA- induced photosensitization and will form the basis for design of more effective PDT regimens for clinical use.